PROJECT SUMMARY Glaucoma, a leading cause of blindness, is managed medically by treating the causal risk factor of increased intraocular pressure (IOP), which is typically observed prior to retina degeneration and loss of visual field. IOP is controlled in the anterior region of the eye, which contains the trabecular meshwork (TM) extracellular matrix, the anatomical pathway for drainage of aqueous humor fluid. Of the ~45 million cases of open angle glaucoma worldwide, ~3% are linked to mutations in myocilin, a protein highly expressed in the TM. Despite considerable research effort over ~20 years, the explicit biological function of myocilin remains unknown. An improved molecular understanding of myocilin in its normal and disease states will change the paradigm for anti-glaucoma therapeutics by enabling agents that target the disease process instead of indirectly controlling IOP. Disease-associated mutations in myocilin are found throughout its sequence, though the best studied are those clustered in its C-terminal olfactomedin (myoc-OLF) domain. Our detailed biophysical and structural characterization of myocilin from the prior grant period lent critical new details and support for the predominant working hypothesis in which mutations localized to myoc-OLF lead to a gain of toxic function: Mutant myocilin aggregates as amyloid within TM cells, and ER-associated degradation is inhibited by an aberrant interaction between myocilin and the endoplasmic-reticulum (ER)-resident chaperone Grp94. This leads to cell death, and the resulting accumulation of TM cell debris is thought to impede fluid outflow from the TM, causing IOP elevation. Continued structure/dysfunction studies of myocilin will not only contribute to our understanding of the TM and glaucoma, but would also broaden our comprehension of the many other OLF domains, which are implicated broadly in physiology and diseases. The objectives of this proposal are to broaden our molecular comprehension of misfolding in myocilin- associated glaucoma as well as provide a path forward for functional studies and the discovery of small molecules that mitigate aberrant myocilin behavior. We will (1) elucidate the structure of native full-length myocilin and compare biophysical and cellular properties of N-terminal variants, (2) clarify the interaction between myocilin and the ER-resident chaperone Grp94 at the molecular level, and (3) implement two high throughput assays. The expected outcomes are (1) support for a loss of adhesion function hypothesis for pathogenic N-terminal myocilin disease variants, (2) expansion of our knowledge of protein conformational disorders, (3) new insights into Grp94 chaperone biology, and (4) novel ligand assays based on the myoc-OLF structure and myoc-OLF/Grp94 interaction for the identification of therapeutic small molecules.